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s-Subunit of the Gs Protein with Microfilaments and Microtubules: Implication during Adrenocorticotropin Stimulation in Rat Adrenal Glomerulosa Cells1
Service of Endocrinology, Departments of Medicine (M.C., N.G.-P.), Anatomy and Cell Biology (M.C., N.G.-P.), and Physiology and Biophysics (M.D.P.), Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4
Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service of Endocrinology, Department of Medicine, Faculty of Medicine, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4. E-mail: n.gallo{at}courrier.usherb.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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s-subunit of the
Gs protein was associated with both structures. Stimulation
of cells with ACTH induced a rapid increase (within 1 min) in the
levels of microfilaments, microtubules, and s associated
with the membrane. In addition, ACTH stimulation of cAMP production was
very sensitive to Ca2+, without any stimulation in
Ca2+-free medium. Under these conditions, actin filaments
were short and formed a dense network. These observations suggest that
the Ca2+-free medium stabilized the actin fibers in such a
way that activation by ACTH failed, further documenting the importance
of microfilaments in cAMP production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the rat adrenal gland, there is little information on the direct
role of the cytoskeleton in the early events of ACTH action, namely
cAMP production, and on a possible association of Gs with
cytoskeleton. The aims of the present study were 1) to study the
influence of cytoskeletal disruption on cAMP production induced by
ACTH, 2) to analyze the time-dependent changes in microfilament and
microtubule organization, and 3) to investigate whether the
s-subunit of the Gs protein could be
associated with microfilaments and microtubules.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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s from New
England Nuclear-DuPont (Mississauga, Canada); anti-q
from Dr. Gilles Guillon (INSERM U-401, Monpellier, France); anti-IgG
antibody from Calbiochem (La Jolla, CA); rhodamine-phalloidin from
Molecular Probes (Eugene, OR); polyvinylidene difluoride membranes and
Immobilon P from Millipore (Bedford, CA); and Vectashield from Vector
Laboratories (Burlingame, CA). All other chemicals were of A grade
purity.
Preparation of glomerulosa cells
The zonae glomerulosa were obtained from adrenal glands of
female Long-Evans rats, weighing 200–250 g, and were isolated
according to the method described in detail previously (22, 23). The
successive steps of zona glomerulosa isolation and cell dissociation
were performed in MEM (supplemented with 100 U/ml penicillin and 100
µg/ml streptomycin). After a 20-min incubation at 37 C in collagenase
(2 mg/ml, 4 capsules/ml) and deoxyribonuclease (25 µg/ml), the cells
were disrupted by gentle aspiration with a sterile 10-ml pipette,
filtered, and centrifuged for 10 min at 100 x g. They
were then resuspended in OPTI-MEM medium supplemented with 2% FBS, 100
U/ml penicillin, and 100 µg/ml streptomycin and plated in 35-mm
tissue culture dishes (for cAMP experiments) or 16-mm multiwell plates
(for steroid measurements) at a density of approximately 1 x
105 cells/multiwell or dish, respectively. The cells were
cultured at 37 C in a humidified atmosphere of 95% air-5%
CO2. The culture medium was changed every day, and the
cells were used after 3 days of culture. At this time, cell density was
approximately 1–3.0 x 105 cells/dish or well in a
multiwell plate.
Incubations for measurement of aldosterone secretion
Before each experiment, the medium of cultured cells was
aspirated, and the cells were washed twice with cold Hanks’ buffered
saline (HBS; 130 mM NaCl, 3.5 mM KCl, 1.8
mM CaCl2, 0.5 mM MgCl2,
2.5 mM NaHCO3, and 5 mM HEPES)
supplemented with 1 g/liter glucose and 0.5% BSA. The cells were
incubated in 1 ml, consisting of 0.9 ml HBS-glucose supplemented with
0.5% BSA-0.1 mg/ml bacitracin and 0.1 ml of stimuli. After a 2-h
incubation at 37 C in an atmosphere of 95% air-5% CO2,
the incubation medium was removed by aspiration and stored at -20 C
until assayed aldosterone in the medium was determined by RIA, using
specific antisera and tritiated steroid as tracer.
cAMP determination
Intracellular cAMP production was determined by measuring the
conversion of [3H]ATP to [3H]cAMP, as
previously described (24). In short, cultured cells were incubated at
37 C in OPTI-MEM culture medium containing 2 µCi/ml
[3H]adenine. After 1 h, the cultures were washed
with HBS buffer and incubated in the same buffer containing 1
mM isobutylmethylxanthine for 15 min at 37 C. The hormones
or drugs were then added to the incubation medium for an additional 15
min at 37 C. The reaction was ended by aspiration of the medium. Cells
were scraped with a rubber policeman, and 100 µl of a cold solution
of ATP and cAMP (5 mM each) were added to the mixture.
Cellular membranes were pelleted at 5000 x g for 15
min, and the supernatants were sequentially chromatographed on Dowex
and alumina columns according to the method of Salomon et
al. (25), allowing the separation of [3H]ATP
nucleotide (primarily [3H]adenine) from
[3H]cAMP. cAMP formation was expressed as: %
conversion = ([3H]cAMP/[3H]cAMP +
[3H]ATP) x 100/15 min.
Membrane preparation
After hormonal stimulation, 3-day cultured cells were washed
twice with HBS buffer and then with 10 mM ice-cold Tris-HCl
buffer (containing 0.5 mM EDTA, 1 mM
MgCl2, 28 mM phenylmethylsulfonylfluoride, 0.04
U/ml aprotinin, and 1 mM benzamidine, pH 8.0). The cells
were then scraped from the substratum with a rubber policeman and
homogenized in a sonicator. Cell extracts were centrifuged at 700
x g, and the resultant supernatant was centrifuged at
30,000 x g to obtain the membrane fraction. The
membrane fraction was resuspended in 50 mM Tris-HCl buffer
(containing 2 mM EDTA, 5 mM MgCl2,
and 250 mM sucrose) and stored at -20 C for subsequent
Western blot assays.
Preparation of microtubules
Preparations enriched in microtubules were extracted from cells
grown in 60-mm petri dishes as described by Solomon (26) with some
modifications. The cells were pretreated with 1 mM taxol
for 2 h before extraction of microtubules. At this concentration,
taxol stabilizes microtubules without promoting polymerization. The
culture medium was then aspirated and replaced with PM2G buffer (0.1
M PIPES, 2 M glycerol, 5 mM
MgCl2, 2 mM EGTA, 0.04 TIU/ml aprotinin, 2
mM phenylmethylsulfonylfluoride, and 1 mM
benzamidine, pH 6.9) containing taxol (1 mM). Cells were
scraped from the substratum with a rubber policeman and transferred to
a 15-ml conical tube and centrifuged at 1,000 x g for
5 min at 37 C. The cell pellet was then extracted with PM2G buffer
containing 1% Nonidet P-40 and 1 mM taxol. After a 15-min
incubation at 37 C, the suspension was centrifuged at 1,000 x
g for 5 min at 37 C. The pellet containing the microtubules
was then solubilized in 125 mM Tris buffer, pH 6.8,
containing 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol and
heated to 100 C for 5 min. After centrifugation at 10,000 x
g for 5 min, the supernatant was stored at -20 C for
subsequent Western blot analysis. For total cell extracts, cells were
grown in 60-mm petri dishes, washed twice with HBS buffer, and
solubilized as described above.
Extraction of microfilaments
Enriched microfilament preparations were extracted from cells
grown in 60-mm petri dishes as described by Phillips et al.
(27). Culture medium was aspirated and changed for HBS buffer. Cells
were scraped from the substratum with a rubber policeman and
transferred in a 15-ml conical tube. Cells were centrifuged at 100
x g for 5 min at room temperature. One hundred milliliters
of Triton solution (1% Triton X-100, 10 mM EGTA, and 0.1
M Tris-HCl, pH 7.4) was added, and the solution was
transferred to 1.5-ml microcentrifuge tubes. After a 10-min incubation
at 0 C, the preparation was centrifuged at 8000 x g
for 4 min at room temperature. Triton-soluble G-actin fraction was
contained in the supernatant. The pellet, which corresponds to the
Triton-insoluble fraction, was solubilized in 2% SDS-2%
2-mercaptoethanol (vol/vol). After a 10-min incubation at 100 C,
F-actin was solubilized. Both fractions were aliquoted and frozen for
subsequent Western blot analysis.
Western blotting
Samples from an equivalent number of cells were compared in each
experiment. Samples were separated on 4–15% SDS-polyacrylamide gels.
Proteins were transferred electrophoretically to polyvinylidene
difluoride membranes. Membranes were blocked with 1% gelatin and
0.05% Tween-20 in Tris-buffered saline (TBS; pH 7.5). After three
washes with TBS-Tween 20 (0.05%), membranes were incubated with
anti-ß tubulin (dilution, 1:250), anti-actin (dilution, 1:100), or
anti-s (dilution, 1:1000) for 3 h at room
temperature, followed by four washes with TBS-Tween-20. Detection was
accomplished using horseradish peroxidase-conjugated antimouse antibody
for actin and tubulin (Amersham) or antirabbit for s and
the enhanced chemiluminescence detection system (Amersham). The
immunoreactive bands were scanned by laser densitometry and expressed
in arbitrary units. Note that the two isoforms of s
proteins were analyzed together.
Immunoprecipitation
Glomerulosa cells in 60-mm petri dishes were washed once and
stimulated with ACTH (100 nM) as indicated at 37 C. The
cells were then washed twice with ice-cold HBS buffer and lysed in TSA
buffer [0.1 M Tris-HCl (pH 8.0), 0.14 M NaCl,
0.025% NaN3, 1% Nonidet P-40, 1% BSA, 1 mM
phenylmethylsulfonylfluoride, 1 mM iodoacetamide, 0.2 U/ml
aprotinin, and 1 mM benzamidine] for 60 min at 4 C.
Lysates were clarified with protein A-Sepharose for 2 h at 22 C,
followed by centrifugation at 200 x g for 1 min. For
immunoprecipitation of actin or tubulin, the lysates were incubated for
2 h with 2 mg/ml monoclonal antibodies at 22 C. Protein
A-Sepharose was added, and incubation was performed overnight at 4 C.
Immunocomplexes were washed five times before electrophoresis on
4–15% SDS-polyacrylamide gels and analysis by immunoblotting.
Immunofluorescence
For immunofluorescence studies, cells were plated on plastic
coverslips (Starsted, St. Laurent, Canada), grown for 3 days, and
treated with appropriate stimuli. For microfilament visualization,
cells were fixed for 1 min with 3% (vol/vol) formaldehyde in PBS
buffer, permeabilized by incubation for 10 min in PBS-0.1% Triton
X-100, and incubated for 20 min at room temperature with 1 U
rhodamine/phalloidin solution. For microtubules, s, and
q detection, cells were fixed for 1 min with 3%
(vol/vol) formaldehyde in 80 mM PIPES (pH 6.5), 5
mM EDTA, and 2 mM MgCl2 and fixed
for an additional 8 min with 3% (vol/vol) formaldehyde in 100
mM sodium borate (pH 11) (18). Cells were then incubated
for 30 min in PBS-0.1% (vol/wt) sodium borohydride; permeabilized by
incubation in PBS-0.2% Triton X-100; incubated overnight at 4 C with
anti-ß tubulin (1:50), anti-s (1:50), or
anti-q (1:50); washed; and further incubated for 60 min
at 37 C with a secondary conjugated anti-IgG antibody coupled with
fluorescein isothiocyanate (FITC). For double immunofluorescence, cells
were fixed and permeabilized as described for microtubule and G protein
detections and processed successively with anti-s or
anti-q and anti-ß tubulin or rhodamine/phalloidin as
described above. After washings, cells were postfixed for 20 min with
3% formaldehyde-PBS and incubated in the presence of 50 mM
NH4Cl for 10 min. The coverslips were then mounted in
Vectashield mounting medium and examined on a Nikon DM 400 microscope
equipped for epifluorescence. B-1E FITC and G-2A rhodamine filters
(Nikon, Melville, NY) were used to visualize images.
Electron microscopy studies
The Triton X-100-insoluble preparations were prepared and fixed
with 2.5% glutaraldehyde, postfixed with 2% osmium, dehydrated, and
embedded in Epon 812. Gold to silver-gray sections were then stained
with uranyl acetate and lead citrate and examined in a Phillips 300
electron microscope (Phillips, Mahway, NJ).
Data analysis
The data are presented as the mean ± SE.
Statistical analyses of the data were performed using one-way ANOVA.
Homogeneity of variance was assessed by Bartlett’s test, and
P values were obtained from Dunnett’s tables. n indicates
the number of experiments; each was performed in triplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To determine how
microtubules may be involved in the activation of adenylyl cyclase, we
measured the dose-dependent effect of colchicine on
AlF-4- and forskolin-stimulated cAMP
production. AlF-4, a nonspecific activator of
all heterotrimeric G proteins (28), and forskolin, which directly
activates the catalytic subunit of adenylyl cyclase (29), stimulated
cAMP production by 16- and 50-fold, respectively (n = 4). Results
from Fig. 1B show that colchicine induced a dose-dependent inhibition
of cAMP production induced by AlF-4 (60.2
± 1.8% decrease; n = 4; P < 0.001 at 10
µM), but did not affect the large increase produced by
forskolin.
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reveals that the addition of cytochalasin B (a
microfilament-disrupting agent) in the incubation medium did not affect
basal cAMP levels, but induced a dose-dependent inhibition of cAMP
accumulation induced by ACTH (55.6 ± 3.1% decrease; n = 4;
P < 0.001 at 10 µM). In contrast to
colchicine, cytochalasin B inhibited both
AlF-4-stimulated (61.3 ± 2.0% decrease;
n = 4; P < 0.001 at 10 µM; Fig. 2B)
and forskolin-stimulated (53.1 ± 4.1% decrease; n = 4;
P < 0.001 at 10 µM) cAMP production
(Fig. 2C).
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, the decrease in cAMP production
induced by cytoskeletal disruption was accompanied by a decrease in
aldosterone secretion. Preincubating glomerulosa cells with 10
µM colchicine or 10 µM cytochalasin B for
30 min did not significantly change the basal output of aldosterone
secretion, whereas stimulation induced by 100 nM ACTH was
drastically decreased by 74% and 80%, respectively. Fluorescence
studies show that these experimental conditions (10 µM,
30 min) were sufficient to achieve a complete disruption of the
microtubular and microfilamentous networks (data not shown).
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). One-minute incubation with ACTH was sufficient to
increase the intensity of actin labeling at the cell periphery (Fig. 3B). This labeling pattern persisted for 10 min of stimulation (Fig. 3C). After 15 min, the intense actin labeling at the membrane
decreased, whereas labeling in the cytoplasm increased (Fig. 3, D and
E). The actin labeling was intense after a 2-h incubation with the
hormone, when an increase in the number and thickness of stress fibers
was evident (Fig. 3F). In contrast, microtubules appeared as long and
thin filaments, loosely distributed throughout the cell (Fig. 4A). Apart from a small increase visible at the
perinuclear region, ACTH treatment did not significantly modify the
microtubular distribution inside the cell (Fig. 4, B–D).
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s protein. Two protocols were used to
evaluate dynamic changes in the levels of microfilaments and
microtubules during ACTH stimulation. In the first set of experiments,
actin-enriched preparations [referred to as the Triton X-100-insoluble
fraction by Phillips et al. (27)] and microtubule-enriched
preparations were analyzed for their respective contents of actin and
tubulin after ACTH stimulation. These values were compared with the
total content of tubulin and actin in whole cell homogenates and with
that specifically associated with cell membrane preparations. Western
blot analyses were performed using antiactin, anti-ß-tubulin, or
anti-s-subunit, as the Gs coupling G protein
is the limiting step in adenylyl cyclase activation. As shown in Fig. 5A, a 1-min incubation with ACTH was sufficient to
induce a huge increase in membrane-associated actin (3.79 ±
0.02-fold increase; n = 3; P < 0.001, compared to
control). This membrane association decreased after 15 min and returned
to basal levels after 2 h of incubation with ACTH (1.69 ±
0.02- and 0.98 ± 0.02-fold increases after 15 min and 2 h,
respectively; n = 3). In contrast, as attested by
immunofluorescence studies (Fig. 3
), the level of polymerized actin
increased after 15-min and 2-h incubations (2.77 ± 0.02- and
3.9 ± 0.2-fold increases, respectively; n = 3;
P < 0.001, compared to control; Fig. 5B, lanes 3 and 4
vs. lane 1). This dynamic redistribution and the increase in
actin content in the Triton-insoluble fraction corresponded to an
increase in polymerization, not in new synthesis of actin, as total
levels of actin in the cell homogenate did not change during the 2-h
incubation with ACTH (Fig. 5C). Figure 5D indicates that ACTH
application induced a time-dependent increase in the level of
microtubules associated with the membrane (3.8 ± 0.2-, 5.9
± 0.2-, and 7.9 ± 0.3-fold increase, for, respectively, lane 2,
1 min; lane 3, 15 min; and lane 4, 2 h; n = 3). All of these
values were significantly increased compared to control values
(P < 0.001). As shown in Fig. 5E, the amount of
polymerized tubulin increased only during the first minutes of
incubation with the hormone (5.25 ± 0.2-, 2.3 ± 0.2-, and
1.99 ± 0.03-fold increase after 15 min and 2 h,
respectively; n = 3). Like actin, the total amount of tubulin in
cell homogenates did not change (Fig. 5F). Simultaneous immunoblots of
the subcellular fractions with anti-s antibody detected
two bands of 45 and 52 kDa (Fig. 6A) and indicated that
s was associated with the Triton-insoluble fraction
(Fig. 6B) and the microtubule-enriched fraction (Fig. 6C). A 1-min
incubation with ACTH strongly increased the amount of s
associated with the membrane (Fig. 6A, lane 2 vs. lane 1),
whereas the amount of s associated with the
microfilament preparation decreased (Fig. 6B). Densitometric analysis
indicated that the level of s associated with the
membrane was increased by 5.1 ± 0.7-fold (n = 3;
P < 0.001 compared to the control; Fig. 6A, lane 2
vs. lane 1) after 1 min and returned to the basal level of
association after 15-min and 2-h incubations (0.97 ± 0.09- and
1.2 ± 0.1-fold increases, respectively; n = 3; Fig. 6A, lanes 3 and 4 vs. lane 1). In contrast, the level of
s associated with microfilaments decreased after 1 min
(0.44 ± 0.004, compared to the control value of 1.0; Fig. 6B, lane 2 vs. lane 1) and increased after 15 min and 2 h
(2.47 ± 0.12- and 2.53 ± 0.09-fold increases, respectively;
Fig. 6B, lanes 3 and 4 vs. lane 1). The transient increase
in s in the membrane preparation was associated with the
increase in actin associated with the membrane (compared Figs. 6A and 5A, lanes 2 vs. lanes 1). In addition, association of
s with microtubules was markedly increased after 15 min
(Fig. 6C, lane 3 vs. lane 1) and was correlated with a
time-dependent increase in the association of microtubules with
membranes (Fig. 5D). To confirm the association of s
with actin and tubulin, cell lysates were immunoprecipitated with
anti-actin and anti-ß-tubulin and then processed for immunoblot
analysis with anti-actin, anti-ß-tubulin, and anti-s
antibodies. The results presented in Fig. 7 confirm that
both isoforms of s were associated with actin and
tubulin. Fifteen-minute incubations with ACTH did not change the amount
of s associated with the microtubules (Fig. 7A), whereas
the amount of s associated with microfilaments increased
(Fig. 7B).
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ss by immunofluorescence. s labeling
appeared as small vesicles (Fig. 8A). The absence of
labeling at the cell periphery may be ascribed to alteration of the
membrane during the permeabilization procedure used to facilitate the
entry of the antibody into the cell. Double staining did not show any
overlap between s labeling and actin or tubulin labeling
(compare Fig. 8A with Figs. 3 and 4). When anti-s was
inactivated by heating at 100 C (Fig. 8B) or when secondary Ig G was
used alone, no labeling was observed. The q labeling
differed from that observed with the s antibody; it
clearly overlapped microfilament labeling (Fig. 8, C and D). Electron
microscopic examination of the Triton-insoluble preparation effectively
confirmed the presence of spherical structures that were closely
associated with microfilaments (Fig. 9, A and B).
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). Immunofluorescence studies revealed that in
control Ca2+-free medium, microfilaments appeared as a
dense network (Fig. 11A). Under these conditions, a
15-min incubation with ACTH did not modify the distribution of actin
filaments as it did in Fig. 3 (Fig. 11B). Moreover, after a 2-h
incubation in a Ca2+-free medium, the network of stress
fibers was completely disrupted, forming short barb-ended filaments
(Fig. 11C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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s-subunit of the Gs protein
is associated with both microtubules and microfilaments. Our results
indicate that ACTH stimulation induces a rapid redistribution of
microfilaments and microtubules at the membrane. This increase enhances
the amount of s associated with membrane, a process that
appears to be important for adenylyl cyclase activation.
Cytoskeleton and cAMP production
Our results show that a 30-min incubation with colchicine
completely blocks cAMP production induced by ACTH, whereas incubation
with cytochalasin B decreases it by 50%. Moreover, our results
indicate that colchicine decreases cAMP production stimulated by
AlF-4 without hindering the effect of
forskolin, indicating that microtubules are essential for
Gs protein activation. These results confirm those of
Feuilloley et al. (3) obtained using human adrenocortical
cells, which demonstrate that colchicine affects ACTH-induced steroid
secretion by acting before cAMP production. A close association between
tubulin and G protein has been demonstrated by the studies of Rasenick
et al. (16, 17, 30, 31, 32). Like G proteins, tubulin binds GTP,
self-assembles, hydrolyzes GTP, and is ADP-ribosylated by cholera and
pertussis toxin (30, 31). More importantly, Roychowdhury et
al. (32) demonstrated that the transfer of GTP from tubulin to
s activates Gs. In addition, cytochalasin B
inhibits stimulation induced by both AlF-4 and
forskolin. These results are in agreement with the observations that
both microtubules and microfilaments may be closely associated with the
enzyme adenylyl cyclase (20) and its activation (21). The fact that
colchicine blocks the fluoroaluminate response, but not the forskolin
response, indicates that microtubules are involved upstream in the
activation of adenylyl cyclase. This supports the observation made in
frog and human adrenals, in which vinblastine had no effect on the
stimulatory action of (Bu)2cAMP on
corticosteroidogenesis (3, 4). However, in Leydig cells (9), leukocytes
(33), and lymphoma cells (34), colchicine increased both basal and
hormone-stimulated cAMP production. Our results indicate that
microtubules and microfilaments are essential for ACTH-stimulated cAMP
production, but they act at different levels; microtubules are
implicated in the activation of Gs protein, whereas
microfilaments are essential for activation of both Gs and
adenylyl cyclase.
ACTH and microfilament and microtubule organization
The rapid increase in membrane-associated actin during ACTH
stimulation supports the importance of the role of microfilaments in
the early events of ACTH action. The initial redistribution of actin at
the membrane is followed by a net increase in actin polymerization,
where stress fiber organization is intensified compared to that in
control cells after a 2-h incubation with ACTH. Western blot analysis
indicates that ACTH not only induces actin redistribution between 1–10
min of incubation, but also promotes polymerization after a 2-h
incubation. The pattern of microfilament rearrangement after
application of ACTH has been shown also by electron microscopic studies
(1, 12, 35). The rapid increase in actin polymerization and association
with the cell membrane has not been previously described in adrenal
cells, but was observed in other cell types, such as blood cells (36, 37). Jennings et al. (36) demonstrated that a 15-sec
stimulation of platelets with thrombin increases the amount of F-actin
by 65% and increases the organization of actin filaments with other
cytoskeletal proteins. In glomerulosa cells, our immunofluorescence
studies show that during the same experimental period, the microtubular
network is not significantly modified, although the amount of tubulin
associated with the membrane did increase. A probable explanation for
these discrepancies may be the alteration of the membrane during the
process of permeabilization used to introduce the antitubulin antibody
in the cell. This is obvious after comparison of Figs. 3 and 8D. In
Fig. 3, the membrane actin network is more evident than in Fig. 8D, where double immunofluorescence was conducted. As reviewed recently
(11), all of our data suggest that microtubules are associated with the
early events of the action of ACTH rather than with the process of
steroidogenesis (8, 38, 39).
Significance of s-associated
cytoskeleton during ACTH stimulation
Immunoblot analyses indicate that s protein is
not only present in the membrane, but is closely associated with
microfilaments and microtubules. One-minute stimulation with ACTH
strongly increases the amounts of s and actin associated
with the membrane, whereas the increased association of
s and tubulin with membrane is maintained for 15 min. We
did not observe any changes in total tubulin or actin content under
ACTH stimulation, indicating that ACTH promotes the polymerization of
actin and tubulin either directly or indirectly without modification of
the total tubulin and actin content.
Immunoblot experiments confirmed that s protein is
associated with both actin and tubulin, whereas immunofluorescence did
not reveal overlapping labeling. The localization of s
as caveolae and the observations obtained from electron microscopy in
which these caveolae are also present in Triton-insoluble preparations
have been previously reported in MDCK kidneys cells (40). As G protein
does not directly bind F-actin, it is likely that cytoskeletal
association of G proteins may be mediated by some actin-associated
proteins. Several newly identified proteins may be good candidates
(41). In this respect, a recent publication reported the presence of a
protein immunodetected around caveolae structures resistant to Triton
X-100 extraction, which was subjected to dynamic movement under ACTH
stimulation (42). This protein of 160 kDa could be an actin-binding
protein, which may link Gs to microfilaments. Moreover,
direct association of s with actin and translocation
from a low speed pellet (actin and associated proteins) to a high speed
pellet (plasma membrane) have been documented during thrombin
stimulation in platelets (43). All of these observations suggest that
s is associated with but not localized on cytoskeletal
elements. Corroborating these results is the morphological evidence
provided by Mattson and Kowal (12). These researchers observed that
ACTH stimulation is accompanied by the formation of several small
vesicles closely associated with microtubules. The observation that
s is associated with both microfilaments and
microtubules is new in the adrenal. However, evidence for direct
control of microfilament polymerization by G proteins has been
described in neutrophils. Särndahl et al. (15)
observed that Gn is associated with the
cytoskeleton (primarily F-actin), but this association decreases or
disappears under fluoroaluminate (AlF-4),
GTP
S, or fMet-Leu-Phe stimulation. Bengtsson et al. (44)
also found that AlF-4 and GTPS are able to
induce an increase in F-actin content, even when phospholipase C
activity is inhibited.
On the other hand, the results presented in Fig. 10 show that cAMP
production is impaired in a low or Ca2+-free medium despite
ACTH binding to its receptor (45). This points out the crucial role of
Ca2+ in the stimulating action of ACTH, although its exact
target is not yet identified. Results presented in Fig. 11 show that a
15-min incubation in a Ca2+-free medium stabilizes actin
fibers in a manner that cannot be activated by ACTH. A 2-h incubation
in a Ca2+-free medium completely disrupts the microfilament
network. Such observations have also been described by Castellino
et al. (46). These researchers have shown that the caldesmon
protein is responsible for this effect. The modification of actin fiber
organization may be correlated with the absence of cAMP production
under ACTH stimulation, supporting a role for microfilaments in the
process of adenylyl cyclase activation.
In summary, the present study demonstrates that both microtubules and
microfilaments are involved in the production of cAMP induced by ACTH
in rat glomerulosa cells. Their role in the transduction signal of ACTH
is due to their close association with s protein. Our
results support the concept 1) that microtubules may be implicated in
the activation of Gs protein, whereas microfilaments are
essential for both Gs and adenylyl cyclase activation; and
2) that rapid dynamic redistribution of both filaments from cytosol to
membranes is responsible for cAMP production.
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Acknowledgments |
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q
protein. We are greatly indebted to Dr. Bernard Schimmer for his
critical review of the manuscript. |
Footnotes |
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2 Recipient of a Scholarship from Les Fonds de La Recherche en
Santé du Québec.
Received June 24, 1996.
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and Gi1.
J Biol Chem 265:1239–1242s/Gi2 proteins define domains on
Gs that interact with tubulin for ß-adrenergic
activation of adenylyl cyclase. J Biol Chem 269:21748–21754 subunits of Gq and G11 G proteins with
actin filaments in WRK1 cells: relation to G
protein-mediated phospholipase C activation. Proc Natl Acad Sci USA 92:8413–8417This article has been cited by other articles:
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) or
presence (•) of 100 nM ACTH (A) or in the presence of 30
mM AlF-4 (
) or 10
µM forskolin (
; B). The [3H]cAMP
concentrations that accumulated were determined and expressed as a
percentage of the total intracellular [3H]ATP, calculated
as described in Materials and Methods. Results are the
mean ± SE of triplicate determinations in one
experiment that is representative of three. When no error bars are
shown, they are contained within the symbols.
), or
100 nM CaCl2 (